Human Extracellular Matrix-1

ABSTRACT

A human ECM-1 polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for stimulating the differentiation in growth of osteoblasts and osteoclasts, which may be used to promote the healing of bone fractures and de novo bone formation, for osteoporosis, for and to promote angiogenesis. Antagonists to the polypeptide of the present invention are also disclosed which may be utilized to treat osteodystrophy, osteohypertrophy, osteoma, osteoblastoma and cancers. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/142,735, filed Jun. 2, 2005, which is a divisional of 10/435,392,filed May 12, 2003, now U.S. Pat. No. 7,199,225, issued Apr. 5, 2007which is a divisional of U.S. application Ser. No. 09/854,549, filed May15, 2001, now U.S. Pat. No. 6,610,829, filed Aug. 26, 2003 which is acontinuation of U.S. application Ser. No. 09/007,105, filed Jan. 14,1998, which claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Nos. 60/035,711, filed Jan. 16, 1997 and 60/050,113, filedJun. 18, 1997, each of which is hereby incorporated herein by referencein its entirety.

STATEMENT UNDER 37 C.F.R. §1.77(b)(5)

This application refers to a “Sequence Listing” listed below, which isprovided as a text document. The document is entitled“PF223C1D2C1_SeqListing.txt” (29,564 bytes, created Feb. 25, 2008, andis hereby incorporated by reference in its entirety herein.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. The polypeptide of the present invention has beenputatively identified as the human homolog of the mouse ExtracellularMatrix-1 protein, sometimes hereinafter referred to as “hECM-1.” Theinvention also relates to inhibiting the action of such polypeptides.

BACKGROUND OF THE INVENTION

The process of embryonic bone formation involves the creation of anextracellular matrix that mineralizes during the course of tissuematuration. This matrix is subject to constant remodeling during thelifetime of an individual, through the combined actions of osteoblastsand osteoclasts. A careful balance of matrix formation and resorptionmust be maintained because perturbations can result in various bonedisorders.

The extracellular matrix of bone consists of two phases, an organicphase and a mineral phase. The organic phase consists primarily of thecollagen type I fibrils that are associated with a number ofnoncollagenous matrix proteins. Interest in the noncollagenous proteinsof the bone has been greatly stimulated since Urist first demonstratedthat demineralized bone extracts could induce ectopic bone formation(Urist, M. R., Science, 150:893-899 (1965)). Noncollagenous proteins ofbone are now believed to be involved in mineralization as well as thelocal regulation of bone cell function (Heinegard, D. and Oldberg, A.,Connective Tissue and Its Heritable Disorders (Royce, P. M. andSteinmann, B., EDS), pages 189-209, Wiley-Liss, New York (1993), and Vonder Mark, K. and Goodman, S., id.). In the past few years, a number ofnoncollagenous proteins of bone have been isolated and characterized;among these are osteocalcin, osteopontin, osteonectin and bonesialoprotein (Heinegard, D. and Oldberg, A., FASEB J., 3:2042-2051(1985)).

A clonal osteogenic cell line (MN7) from bone marrow stroma of the adultmouse has been established (Mathieu, E., et al., Calcif. Tissue Int.,50:362-371 (1992)). These cells, under appropriate conditions, undergotypical osteoblastic differentiation in vitro and are able to form amineralized extracellular matrix (athieu, E. and Merregaert, J., J. BoneMiner. Res., 9:183-192 (1994)).

A cDNA coding for a novel secretory protein of mouse (p85), has beencloned, characterized and genetically mapped (Bhalerao, J., et al., J.Biol. Chem., 270 (27):16385-16394 (1995)). The full-length cDNA containsan open reading frame of 1677 bp encoding a protein of 559 amino acids.The clone contains a hydrophobic signal peptide characteristic of asecreted protein. The message of 1.9 kb is expressed in various tissues,such as liver, heart, lungs, etc., whereas a splice variant was presentin embryonic cartilage in skin. This gene p85, called Ecm1 forextracellular matrix protein 1, maps on chromosome 3 of mouse in aregion containing several loci involved in skin development disorders.

The polypeptide of the present invention has highest amino acid sequencehomology to growth factor Ecm1.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptide of the present invention is ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding a polypeptide of thepresent invention including mRNAs, cDNAs, genomic DNAs as well asanalogs and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

In accordance with another aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the DNA contained in ATCC Deposit No. 97302.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, tostimulate osteoblast and osteoclast differentiation and growth, whichmay be utilized to treat bone disorders and promote bone formation forhealing of bone fractures and treatment of osteoporosis and osteogenesisimperfecta, and to stimulate angiogenesis, which may be utilized torevascularize injured tissue.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with another aspect of the present invention, there areprovided hECM-1 agonists which mimic hECM-1 and bind to the hECM-1receptors and antagonists against such polypeptides, which may be usedto inhibit the action of such polypeptides. The agonists may be employedto treat disease conditions related to an underexpression of the ECM-1polypeptide and the antagonists may be employed to treat diseaseconditions related to an overexpression of such polypeptide. Suchdisease conditions include, for example, osteodystrophy,osteohypertrophy, osteoma, osteopetrosis, osteoporosis, osteoblastoma,and cancer.

In accordance with yet a further aspect of the present invention, thereis also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to a nucleic acidsequence of the present invention.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to mutations in the nucleic acid sequences encodinga polypeptide of the present invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, for example, synthesis of DNA andmanufacture of DNA vectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A-F are an illustration of the cDNA (SEQ ID NO:1) andcorresponding deduced amino acid sequence (SEQ ID NO:2) of thepolypeptide of the present invention. The underlined portion isindicative of a putative leader sequence. Sequencing was performed usinga 373 automated DNA sequencer (Applied Biosystems, Inc.).

FIG. 2 is an amino acid sequence comparison between the polypeptide ofthe present invention (SEQ ID NO:2) (top line) and murine Ecm1 (SEQ IDNO:7) (bottom line).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIGS. 1A-F (SEQ IDNO:2).

The polynucleotide of this invention was discovered in a cDNA libraryderived from a human tumor pancreas. It is structurally related to themurine Ecm1. It contains an open reading frame encoding a protein of 540amino acid residues of which the first 19 amino acids residues are theputative leader sequence (underlined in FIGS. 1A-F) such that the matureprotein comprises 521 amino acids (amino acids 20-540 in FIGS. 1A-F). Ascan be seen in FIG. 2, the protein exhibits the highest degree ofhomology to murine Ecm1 at the amino acid level with 69.4% identity and81.3% similarity over the entire amino acid stretch. The gene of thepresent invention exhibits the highest degree of homology at thenucleotide level also to murine Ecm1 with 80% identity and 80%similarity over the entire nucleotide sequence.

In accordance with another aspect of the present invention there areprovided isolated polynucleotides encoding a mature polypeptideexpressed by the DNA contained in ATCC Deposit No. 97302, deposited withthe American Type Culture Collection, University Boulevard, Manassas,Va. 20110-2209, USA, on Sep. 25, 1995. The deposited material is aplasmid that contains the full-length hECM-1 cDNA inserted into apBluescript SK(−) vector (Stratagene, La Jolla, Calif.).

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. These deposits are provided merely as convenience to those ofskill in the art and are not an admission that a deposit is requiredunder 35 U.S.C. §112. The sequence of the polynucleotides contained inthe deposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted. References to “polynucleotides” throughoutthis specification includes the DNA of the deposit referred to above.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIGS. 1A-F (SEQ ID NO:1) or may be adifferent coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same maturepolypeptide as the DNA of FIGS. 1A-F (SEQ ID NO:1).

The polynucleotide which encodes for the mature polypeptide of FIGS.1A-F (SEQ ID NO:2) may include, but is not limited to: only the codingsequence for the mature polypeptide; the coding sequence for the maturepolypeptide and additional coding sequence such as a leader or secretorysequence or a proprotein sequence; the coding sequence for the maturepolypeptide (and optionally additional coding sequence) and non-codingsequence, such as introns or non-coding sequence 5′ and/or 3′ of thecoding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIGS. 1A-F (SEQ ID NO:2). The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIGS. 1A-F (SEQ ID NO:2) as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIGS. 1A-F (SEQ ID NO:2).Such nucleotide variants include deletion variants, substitutionvariants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A-F (SEQ ID NO:1). As is known in the art, an allelicvariant is an alternate form of a polynucleotide sequence which may havea substitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promoter regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 85%,preferably at least 90%, and more preferably at least 95%, 96%, 97%,98%, or 99% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term “stringent conditions” means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich either retain substantially the same biological function oractivity as the mature polypeptide encoded by the cDNAs of FIGS. 1A-F(SEQ ID NO:1).

Alternatively, the polynucleotide may have at least 20 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridizeto a polynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast an 85% identity, preferably at least a 90% identity and morepreferably at least a 95%, 96%, 97%, 98%, or 99% identity to apolynucleotide which encodes the polypeptide of SEQ ID NO:2 andpolynucleotides complementary thereto, as well as portions thereof,which portions have at least 30 consecutive bases and more preferably atleast 50 consecutive bases and to polypeptides encoded by suchpolynucleotides.

The present invention further relates to a polypeptide which has thededuced amino acid sequence of FIGS. 1A-F (SEQ ID NO:2), as well asfragments, analogs and derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIGS. 1A-F (SEQ ID NO:2), means a polypeptide whichretains essentially the same biological function or activity as suchpolypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIGS. 1A-F (SEQID NO:2) may be (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of hECM-1.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet-forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of hECM-1.

Certain preferred regions in these regards include, but are not limitedto, regions of the aforementioned types identified by analysis of theamino acid sequence set out in FIGS. 1A-F. Such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions andcoil-regions, Chou-Fasman alpha-regions, beta-regions and turn-regions,Kyte-Doolittle hydrophilic regions and hydrophilic regions, Eisenbergalpha and beta amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf high antigenic indexregions.

Particularly preferred polypeptides comprise the entire amino acidsequence shown in FIGS. 1A-F (SEQ ID NO:2) except the amino terminalmethionine. Accordingly, the present invention provides a polypeptidecomprising an amino acid sequence at least 95% identical to the aminoacid sequence shown in FIGS. 1A-F lacking the amino terminal methionine.Polynucleotides encoding such polypeptides are also provided.

Also forming part of the invention are polypeptides comprising the aminoacid sequence of a splice variant of hECM-1, sometimes hereinafter“hECM-1-SV1”. hECM-1-SV1 is missing 375 nucleotides which code for 125amino acids. The region of hECM-1-SV1 missing from the hECM-1 cDNA isshown in FIGS. 1A-F as a boxed region containing nucleotides 812-1186.The hECM-1-SV1 nucleotide sequence is set out as SEQ ID NO:8. Thecorresponding hECM-1 amino acid sequence is shown as SEQ ID NO:9.Accordingly, the invention provides polypeptides comprising an aminoacid sequence at least 95% identical to the amino acid sequence ofhECM-1-SV1. Polynucleotides encoding such polypeptides are alsoprovided.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 80% similarity (preferably at least 80% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95%, 96%, 97%, 98%, and99% similarity (still more preferably at least 95%, 96%, 97%, 98%, or99% identity) to the polypeptide of SEQ ID NO:2 and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

Human ECM-1 is thought to stimulate osteogenesis and angiogenesis(particularly in embryonic development). Therefore, given the activitiesmodulated by hECM-1, it is readily apparent that a substantially altered(increased or decreased) level of expression of hECM-1 in an individualcompared to the standard or “normal” level produces pathologicalconditions such as those described below. It will also be appreciated byone of ordinary skill that, since the hECM-1 protein of the invention istranslated with a leader peptide suitable for secretion of the matureprotein from the cells which express hECM-1, when hECM-1 protein(particularly the mature form) is added from an exogenous source tocells, tissues or the body of an individual, the protein will exert itsmodulating activities on any of its target cells of that individual.Therefore, it will be appreciated that conditions caused by a decreasein the standard or normal level of hECM-1 activity in an individual,particularly disorders relating to fetal development, osteogenesis andangiogenesis, can be treated be administration of hECM-1 protein. Thus,the invention also provides a method of treatment of an individual inneed of an increased level of hECM-1 activity comprising administeringto such an individual a pharmaceutical composition comprising an amountof an isolated hECM-1 polypeptide of the invention, particularly amature form of the hECM-1 protein of the invention, effective toincrease the hECM-1 activity level in such an individual.

More in particular, the hECM-1 gene and gene product of the presentinvention may be employed to promote osteoblast and osteoclastdifferentiation and growth, as well as mineralization of bone.Accordingly, hECM-1 may be employed to promote bone growth, to treatosteoporosis, osteogenesis imperfecta and facilitate the healing offractures.

hECM-1 gene and gene product of the present invention may also beemployed to promote angiogenesis, especially in early fetal developmentand, for example, in revascularization of transplanted or injuredtissue, for example, to stimulate the growth of transplanted tissuewhere coronary bypass surgery is performed. An hECM-1 polypeptide mayalso be employed to stimulate wound healing, particularly tore-vascularize damaged tissues or where new capillary angiogenesis isdesired. An hECM-1 polypeptide may be employed to treat full-thicknesswounds such as dermal ulcers, including pressure sores, venous ulcers,and diabetic ulcers. In addition, hECM-1 polypeptides may be employed totreat full-thickness burns and injuries where a skin graft or flap isused to repair such burns and injuries and also may be employed for usein plastic surgery, for example, for the repair of lacerations fromtrauma and cuts in association with surgery. hECM-1 may also be used totreat ischemia.

Along these same lines, an hECM-1 polypeptide may be employed to inducegrowth of damaged bone, periodontium or ligament tissue.Neo-vascularization is very important in fracture repair, as evidencedby blood vessel development at the site of bone injuries. An hECM-1polypeptide may also be employed for regeneration supporting tissues ofthe teeth, including cementum and periodontal ligament, that have beendamaged by disease and trauma.

Since angiogenesis is important in keeping wounds clean andnon-infected, an hECM-1 polypeptide may be employed in association withsurgery and following the repair of cuts. It may also be employed forthe treatment of abdominal wounds where there is a high risk ofinfection.

An hECM-1 polypeptide may be employed for the promotion ofendothelialization in vascular graft surgery. In the case of vasculargrafts using either transplanted or synthetic material, an hECM-1polypeptide can be applied to the surface of the graft or at thejunction to promote the growth of vascular endothelial cells. An hECM-1polypeptide may also be employed to repair damage of myocardial tissueas the result of myocardial infarction and may also be employed torepair the cardiac vascular system after ischemia. Further, an hECM-1polypeptide may also be employed to treat damaged vascular tissue as aresult of coronary artery disease and peripheral and CNS vasculardisease.

An hECM polypeptide may also be employed for vascular tissue repair, forexample, that required during arteriosclerosis and following balloonangioplasty where vascular tissues are damaged and may also be employedto coat artificial prostheses or natural organs which are to betransplanted in the body to minimize rejection of the transplantedmaterial and to stimulate vascularization of the transplanted materials.

An hECM-1 polypeptide may be employed as a vascularizing agent toimpregnate or coat implant materials for the timed release ofpharmaceutical agents, as for example, when such implants are employedsubcutaneously.

The polynucleotides and polypeptides of the present invention may alsobe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease.

This invention provides a method for identification of the receptor forhECM-1. The gene encoding the receptor can be identified by numerousmethods known to those of skill in the art, for example, ligand panningand FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2),Chapter 5, (1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to hECM-1, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to hECM-1.Transfected cells which are grown on glass slides are exposed to labeledhECM-1, which may be labeled by a variety of means including iodinationor inclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor. As an alternative approach for receptoridentification, labeled hECM-1 can be photoaffinity linked with cellmembrane or extract preparations that express the receptor molecule.Cross-linked material is resolved by PAGE and exposed to X-ray film. Thelabeled complex containing the ligand-receptor can be excised, resolvedinto peptide fragments, and subjected to protein microsequencing. Theamino acid sequence obtained from microsequencing would be used todesign a set of degenerate oligonucleotide probes to screen a cDNAlibrary to identify the gene encoding the putative receptor.

This invention provides a method of screening compounds to identifythose which enhance (agonists) or block (antagonists) interaction ofhECM-1 with its receptor. As an example, a mammalian cell or membranepreparation expressing the hECM-1 receptor is incubated with labeledhECM-1 in the presence of the compound. The ability of the compound toenhance or block this interaction could then be measured. Potentialantagonists include an antibody, or in some cases, an oligopeptide,which are specific to an epitope of the hECM-1 polypeptide.Alternatively, a potential antagonist may be a closely related proteinwhich binds to the receptor sites, however, they are inactive forms ofthe polypeptide and thereby prevent the action of hECM-1 since receptorsites are occupied.

Another potential antagonist is an antisense construct prepared usingantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5′ coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix—see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of hECM-1. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into hECM-1 polypeptide (Antisense—Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of hECM-1.

Potential antagonists include a small molecule which binds to andoccupies the active site of the polypeptide thereby making itinaccessible to substrate such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

The agonists and antagonists may be employed to augment of reduce thebiological effects of the polypeptide of the present invention whereappropriate in the treatment of osteodystrophy, osteohypertrophy,osteoblastoma, osteopertrosis, osteoporosis, osteoma and osteoblastoma.Antagonists will be particularly useful in the treatment of cancer byinhibiting the angiogenesis of tumors. It is significant in this regardthat the deposited cDNA was isolated from cancerous tissue and thathECM-1 is found by immunohistochemical staining in several canceroustissues.

hECM-1 polypeptide stimulation of neovascular activity may be asignificant factor in allowing various cancers to become invasive ormetastasize. In addition to invasive cancer, various other animaldisorders involve abnormally high neovascular activity. In such cases,antagonists to hECM-1 stimulation of neovascularization may be generallyuseful in controlling the progression of such conditions.

Potential antagonists include antibodies which bind to hECM-1polypeptides and effectively eliminate or reduce hECM-1 function.Alternatively, a potential antagonist may be a closely related proteinwhich binds to hECM-1 receptors, but are inactive forms of thepolypeptide, thereby preventing the action of a hECM-1 polypeptide.Examples of these antagonists include a negative dominant mutant of ahECM-1 polypeptide, for example, the polypeptide may be mutated suchthat biological activity is not retained. By binding its receptor, thesenegative mutants are “dominant” in that it brings about a loss of hECM-1polypeptide activity, even though some wild-type hECM-1 polypeptides areproduced from the other allele.

Another potential hECM-1 antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, oligonucleotides complementary to splice junctions areparticularly effective antagonists of the expressed product, by havingdisrupted the mRNA processing events necessary for an active product. ADNA oligonucleotide is designed to be complementary to a region of thegene involved in transcription (triple helix—see Lee et al., Nucl. AcidsRes. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan etal., Science 251:1360 (1991)), thereby preventing transcription and theproduction of a hECM-1 polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the hECM-1 polypeptide (Antisense—Okano, J. Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of hECM-1.

Potential hECM-1 antagonists also include small-molecules which bind toand occupy the active site of the polypeptide thereby making thecatalytic site inaccessible to substrate such that normal biologicalactivity is prevented. Examples of small molecules include, but are notlimited to, small peptides or peptide-like molecules. Small moleculescan also act to block transcription and translation by binding to DNA orRNA such that transcription and translation factors cannot bind. Forsmall molecules to function in this system they must be small enough topass through the cell and nuclear membranes.

The antagonists may also be used to treat inflammation caused byincreased vascular permeability. In addition to these disorders, theantagonists may also be used to treat diabetic retinopathy, rheumatoidarthritis and psoriasis.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The polypeptides of the present invention, and agonists and antagonists,may be employed in combination with a suitable pharmaceutical carrier.Such compositions comprise a therapeutically effective amount of thepolypeptide, agonists or antagonist, and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention, or agonists or antagonists, maybe employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, parenterally, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

The hECM-1 polypeptides and agonists and antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the gene of the presentinvention as a diagnostic. Detection of a mutated form of the gene willallow a diagnosis of a disease or a susceptibility to a disease whichresults from underexpression of hECM-1.

Individuals carrying mutations in the gene of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, including but notlimited to blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al, Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encodinghECM-1 can be used to identify and analyze mutations. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled RNA oralternatively, radiolabeled antisense DNA sequences. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNase Adigestion or by differences in melting temperatures.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al, PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of the polypeptide of the present invention in varioustissues since an over-expression of the proteins compared to normalcontrol tissue samples can detect the presence of bone disorders, forexample, osteoporosis. Assays used to detect levels of the polypeptideof the present invention in a sample derived from a host are well-knownto those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis and preferably anELISA assay. An ELISA assay initially comprises preparing an antibodyspecific to the hECM-1 antigen, preferably a monoclonal antibody. Inaddition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or in this example a horseradishperoxidase enzyme. A sample is now removed from a host and incubated ona solid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attached to any of the polypeptide of thepresent invention attached to the polystyrene dish. All unboundmonoclonal antibody is washed out with buffer. The reporter antibodylinked to horseradish peroxidase is now placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound to thepolypeptide of the present invention. Unattached reporter antibody isthen washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of the polypeptide of the present invention present in agiven volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to thepolypeptide of the present invention are attached to a solid support andlabeled hECM-1 and a sample derived from the host are passed over thesolid support and the amount of label detected attached to the solidsupport can be correlated to a quantity of the polypeptide of thepresent invention in the sample.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA having at least 50 or60 bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

The hECM-1 gene of the present invention has been mapped to humanchromosome 1q21. Once a sequence has been mapped to a precisechromosomal location, the physical position of the sequence on thechromosome can be correlated with genetic map data. Such data are found,for example, in V. McKusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Therelationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysis(coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μL of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLES Example 1 Cloning and Expression of hECM-1 Using theBaculovirus Expression System

The DNA sequence encoding the full length hECM-1 protein, A-TCC # 97302,was amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ CGGGATCCGCCATCATGGGGACCACAGCCAG 3′(SEQ ID NO:3) and contains a BamHI restriction enzyme site (in bold)followed by nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) just in front of the initiation codon for translation“ATG” (underlined).

The 3′ primer has the sequence 5′ GCTCTAGATCCAAGAGGTGTTTAGTG 3′ (SEQ IDNO:4) and contains the cleavage site for the restriction endonucleaseXbaI and 18 nucleotides complementary to the 3′ non-translated sequence.The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with the endonucleases BamHI andXbaI and then purified again on a 1% agarose gel. This fragment isdesignated F2.

The vector pA2 (modification of pVL941 vector, discussed below) is usedfor the expression of the hECM-1 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2 such as pAc373, pVL941and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology, 170:31-39).

The plasmid was digested with the restriction enzymes BamHI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA was then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E. coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBachECM-1) with the hECM-1 geneusing the enzymes BamHI and XbaI. The sequence of the cloned fragmentwas confirmed by DNA sequencing.

5 μg of the plasmid pBachECM-1 was co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BACULOGOLD™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BACULOGOLD™ virus DNA and 5 mg of the plasmid pBachECM-1 weremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the virus was added to the cellsand blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-hECM-1 at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5μCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labeled proteins visualized bySDS-PAGE and autoradiography.

The supernatant (1000 ml) containing baculovirus expressed hECM-1 wasapplied without dilution to an HS-50 column (1.0×10 cm, PerseptiveBiosystems) equilibrated with 0.02M Bis-Tris, pH 6.0, containing 10%glycerol and 0.02 M NaCl (Solvent A) at a flow rate of 8 ml/min.Proteins were then eluted using a gradient from 10% Solvent B (Solvent Acontaining 2 M NaCl) to 30% B. The pooled peak contained 11 mg of hECM-1having a purity of greater than 80%.

Example 2 Expression of Recombinant HECM-1 in COS Cells

The expression of plasmid, hECM-1 HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, an SV40 intron and polyadenylation site. A DNAfragment encoding the entire hECM-1 precursor and a HA tag fused inframe to its 3′ end was cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (I. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell37:767, (1984)). The infusion of HA tag to the target protein allowseasy detection of the recombinant protein with an antibody thatrecognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding hECM-1, ATCC # 97302, was constructed by PCRusing two primers: the 5′ primer 5′ GCGCGGATCCACCATGGGGACCACAGCCAGA 3′(SEQ ID NO:5) contains a BamHI site followed by 18 nucleotides of hECM-1coding sequence starting. front the initiation codon; the 3′ sequence 5′GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTATTCTTCCTTGGGC TC 3′ (SEQ IDNO:6) contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 15 nucleotides of the hECM-1 coding sequence(not including the stop codon). Therefore, the PCR product contains aBamHI site, hECM-1 coding sequence followed by HA tag fused in frame, atranslation termination stop codon next to the HA tag, and an XbaI site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digestedwith BamHI and XbaI restriction enzyme and ligated. The ligation mixturewas transformed into E. coli strain SURE (available from StratageneCloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037)the transformed culture was plated on ampicillin media plates andresistant colonies were selected. Plasmid DNA was isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant hECM-1, COScells were transfected with the expression vector by DEAE-DEXTRAN method(J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989)). The expression of thehECM-1 HA protein was detected by radio labeling and immunoprecipitationmethod (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, (1988)). Cells were labeled for 8 hours with³⁵S-cysteine two days post transfection. Culture media was thencollected and cells were lysed with detergent (RIPA buffer (150 mM NaCl,1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I.et al., Id. 37:767 (1984)). Both cell lysate and culture media wereprecipitated with an HA specific monoclonal antibody. Proteinsprecipitated were analyzed on 15% SDS-PAGE gels.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

Example 3 Immunohistochemical Detection of hECM-1

Immunohistochemistry using a purified polyclonal antibody (269TP)generated in the rabbit against the human Ecm1 protein was performed onparaffin section of archival formalin fixed material. The materialincluded an adenocarcinoma of the lung, a squamous cell carcinoma of theskin and the esophagus, a basal cell carcinoma of the skin, achondrosarcoma of the femur, an adenoma of the parathyroid andhyperplastic parathyroid tissue, an oat cell carcinoma of the lung, aductal carcinoma of the breast and placenta and fetus with a gestationalperiod of 22 weeks. All slides contained tumoral tissue and adjacentnormal tissue. Prior to the immunohistochemistry the paraffin slideswere deparaffinised and the endogenous peroxidase was abolished. Afterrinsing the slides in PBS the slides were preincubated with normal goatserum. In the first step the slides were incubated with primary Ecm1antibody in a dilution of 1/20 for 30 minutes at room temperature. Afterrinsing in PBS the slides were then incubated with the second antibody(goat anti-rabbit from DAKA) for 30 minutes and after thorough rinsingin PBS they were labeled in a third step with the peroxidase conjugatedstreptavidin-biotin complex (DAKO) after which the slides were rinsedonce more in PBS. The chromagen used was DAB and a hematoxylincounterstain was performed.

Results

In the chondrosarcoma some chondrocytes in the lesion show faintimmunoreactivity in the cytoplasm. In the adenocarcinoma of the lung andin the fetal lung tissue there was also cytoplasmic immunoreactivity insome chondrocytes in the bronchial wall. In the adult there was alsosome reactivity in serous epithelial cells from the peribronchialglands. In all the other lung structures and in the tumour there was noimmunoreactivity. In some of the most superficial cells of the normalsquamous epithelium and in the squamous carcinomas in a the skin as wellas in the esophagus moderate cytoplasmic immunoreactivity was seen.There is no reactivity in the surrounding normal tissues. There was noimmunoreactivity in the hyperplastic parathyroid and in the adenoma ofthe parathyroid. In the breast carcinoma the neoplastic cells showedcytoplasmic immunoreactivity. There was also immunostaining inmyoepithelial cells and in adipocytes. There was no immunoreactivity inthe basal cell carcinoma from the skin and in the oatcell carcinoma.With the exception of the chondrocytes in the bronchial wall there wasno immunoreactivity in the fetal organs or the placenta and cord.

Example 4 HUVEC Proliferation Assay

HUVEC were seeded in medium supplemented with the rhEcm1 protein toconcentrations of 100 ng/ml, 10 ng/ml and 1 ng/ml. As a negative controlHUVEC were seeded in medium supplemented with the rhEcm1 proteinsuspension buffer. The proliferation of HUVEC was assessed 18 hourslater with Alamar blue reagent (Johnson et al., 1995). The rhEcm1protein was found to stimulate endothelial cell proliferation in adose-dependent way. A strong effect was observed with a concentration of100 ng/ml. The smallest significant effect could still be detected at 10ng/ml (p<0.01, paired t-test).

1. An isolated nucleic acid molecule comprising a polynucleotide havinga nucleotide sequence at least 95% identical to a sequence selected fromthe group consisting of: (a) a nucleotide sequence encoding the hECM-1polypeptide consisting of amino acid residues 1 to 540 of SEQ ID NO:2;(b) a nucleotide sequence encoding the hECM-1 polypeptide consisting ofamino acid residues 2 to 540 of SEQ ID NO:2; (c) a nucleotide sequenceencoding the HECM-1 polypeptide consisting of amino acid residues 20 to540 of SEQ ID NO:2; (d) a nucleotide sequence encoding the cytostatin IIpolypeptide consisting of the full-length polypeptide encoded by thecDNA contained in ATCC™ Deposit Number 97302; (e) a nucleotide sequenceencoding the hECM-1 polypeptide consisting of the full-lengthpolypeptide minus the N-terminal methionine encoded by the cDNAcontained in ATCC™ Deposit Number 97302; (f) a nucleotide sequenceencoding the hECM-1 polypeptide consisting of the mature form of thepolypeptide encoded by the cDNA contained in ATCC™ Deposit Number 97302;and (g) a nucleotide sequence encoding the hECM-1-SV1 polypeptideconsisting of amino acid residues 1 to 415 of SEQ ID NO:9.
 2. A methodfor making a recombinant vector comprising inserting an isolated nucleicacid molecule of claim 1 into a vector.
 3. A recombinant vector producedby the method of claim
 2. 4. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 3 into a hostcell.
 5. A recombinant host cell produced by the method of claim
 4. 6. Arecombinant method of producing a hECM-1 polypeptide, comprisingculturing the recombinant host cell of claim 5 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 7.An isolated hECM-1 polypeptide comprising an amino acid sequence atleast 95% identical to a sequence selected from the group consisting of:(a) the amino acid sequence of the hECM-1 polypeptide consisting ofamino acid residues 1 to 540 of SEQ ID NO:2; (b) the amino acid sequenceof the hECM-1 polypeptide consisting of amino acid residues 2 to 540 ofSEQ ID NO:2; (c) the amino acid sequence of the hECM-1 polypeptideconsisting of amino acid residues 20 to 540 of SEQ ID NO:2; (d) theamino acid sequence of the hECM-1 polypeptide consisting of thefull-length polypeptide encoded by the cDNA contained in ATCC™ DepositNumber 97302; (e) the amino acid sequence of the hECM-1 polypeptideconsisting of the full-length polypeptide minus the N-terminalmethionine encoded by the cDNA contained in ATCC™ Deposit Number 97302;(f) the amino acid sequence of the hECM-1 polypeptide consisting of themature form of the polypeptide encoded by the cDNA contained in ATCC™Deposit Number 97302; (g) the amino acid sequence of the hECM-1-SV1polypeptide consisting of amino acid residues 1 to 415 of SEQ ID NO:9.8. The polypeptide of claim 7, wherein said polypeptide comprises aheterologous polypeptide.
 9. The polypeptide of claim 7, wherein saidpolypeptide is glycosylated.
 10. A composition comprising thepolypeptide of claim 7 and a pharmaceutically acceptable carrier.
 11. Anisolated polypeptide produced by the method comprising: (a) expressingthe polypeptide of claim 7 in a recombinant host cell comprising anucleic acid molecule encoding said polypeptide; and (b) recovering saidpolypeptide.
 12. An isolated antibody or fragment thereof thatspecifically binds to a hECM-1 polypeptide of claim
 7. 13. An agonist tothe polypeptide of claim
 7. 14. An antagonist to the polypeptide ofclaim
 7. 15. A method for the treatment of a patient having need ofhECM-1 comprising: administering to the patient a therapeuticallyeffective amount of the polypeptide of claim
 7. 16. A method ofstimulating angiogenesis in a patient comprising administering to thepatient the polypeptide of claim
 7. 17. A method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising: (a) determining a mutation in thepolynucleotide of claim
 1. 18. A method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising: (a) determining the presence or amount of expression of thehECM-1 polypeptide using the antibody or fragment thereof of claim 12;and (b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or amount of expression ofthe hECM-1 polypeptide.
 19. A method of inhibiting angiogenesis in apatient comprising administering to said subject the antibody of claim12.